Antibiotic development is a major area of unmet medical need. Nowhere is this need greater than in the case of tuberculosis (TB), the leading bacterial cause of deaths worldwide. A major roadblock to control of the TB pandemic is the lack of drugs active against both replicating and non-replicating forms of its causative agent, Mycobacterium tuberculosis (Mtb). Knowledge of new targets against both replicating and non-replicating Mtb is thus a major area of unmet scientific need. One potent, but understudied, class of compounds active against both replicating and non-replicating Mtb is the reactive nitrogen intermediates (RNI). In vitro, RNI kill Mtb with a molar potency greater than that of many currently used TB drugs. Knowledge of the antimycobacterial mechanism(s) of RNI thus represents a conceptual blueprint to potential targets in replicating and non-replicating Mtb alike. In previous work, we identified proteomic targets of RNI-mediated damage in Mtb. This work was motivated by the view that targets of RNI-mediated damage represented potential drug targets whose inhibition by compounds unrelated to RNI would mimic RNI's mycobactericidal activity and resist Mtb's RNI defenses. Surprisingly, this work selectively identified enzymes of Mtb's intermediary metabolism as a major class of potential drug targets, not targeted by existing TB drugs. Here, we propose to apply a novel mass spectrometry-based metabolomics tool that will not only help define the antimycobacterial actions of RNI against both replicating and non-replicating Mtb but more broadly expand knowledge of Mtb's metabolic network at the biochemically global level of metabolites. To do so, we will deduce the MOA or targets of pharmacologic compounds using unbiased, comparisons of RNI-mediated killing on the Mtb metabolome across and between a variety of settings (analogous to the already proven "compendium" approach used in genomics). As proof-of-concept validation, we present Preliminary Data that affirm the conceptual merit of metabolite profiling (using Mtb's free amino acid pools) as well as the technical feasibility of global metabolite profiling of Mtb. We specifically seek to identify a "universal" metabolomic signature of RNI that would explain their exquisite potency against replicating and non-replicating Mtb alike. Such knowledge would provide a fresh lens on new drug targets that might augment or synergize with natural immunity. From a broader point of view, this work will not only advance efforts to identify new potential drug targets in replicating and non- replicating Mtb but also lay the groundwork for future studies of antimycobacterial compounds with undefined MOA. PUBLIC HEALTH RELEVANCE: Antibiotic development is a major area of unmet medical need. Nowhere is this need greater than in the case of tuberculosis (TB), the leading bacterial cause of deaths worldwide. A major roadblock to control of the TB pandemic is the lack of drugs active against both replicating and non-replicating forms Mycobacterium tuberculosis (Mtb), the causative agent of TB. Knowledge of new targets against both replicating and non-replicating Mtb thus addresses a major unmet scientific need of the current pandemic.